Skeletal muscle arises after the induction of the mesoderm. After differentiation of the mesoderm into dorsal, intermediate, and lateral mesoderm, the dorsal mesodermal mesenchyme differentiates to form myotomes which, in turn, differentiate to give rise to the myogenic precursor cells which ultimately form skeletal muscle. Unlike the myogenic precursor cells of the heart, the skeletal muscle precursors fuse side-to-side to form unbranched, multinucleated myofibers. Some of the skeletal myogenic precursor cells do not differentiate and fuse into myocytes (also called myofibers) but, rather, attach to the outside of the plasmalemma of the myocytes. These cells participate in muscle growth during maturation and typically thereafter will remain, throughout adulthood, as largely undifferentiated, quiescent skeletal muscle “satellite cells.” Upon injury of a skeletal muscle, these satellite cells are revealed to be myogenic precursor cells, or muscle “stem cells,” which proliferate and differentiate, again by fusion, into new and functional skeletal muscle. Even after injury, some of the proliferated satellite cells remain undifferentiated and attach to the newly formed myofibers. Thus, the satellite cells of skeletal muscle provide a constant and renewable source myogenic precursor cells which allows for skeletal muscle repair and regeneration throughout mammalian life.
The proliferation and differentiation of skeletal muscle satellite cells has been extensively studied in vitro. For example, a simple saline extract of skeletal muscle has been shown to cause satellite cells to proliferate in culture (Bischoff (1989) in Myoblast Transfer Therapy, Griggs and Karpati, eds., pp. 147–158). Similarly, it has been shown that chick embryo extract or the conditioned medium of differentiated myotubes from young mice exhibits a strong mitogenic effect on satellite cells, but that conditioned medium from older murine myotubes has a lesser effect (Mezzogiorno et al. (1993) Mech. Ageing & Develop. 70:35–44). In addition, a number of hormones and growth factors have ben found to enhance satellite cell proliferation, including FGF, PDGF, ACTH, LIF, IGF (Bischoff (1989); Mezzogiorno et al. (1993)) and HGF (Tatsumi et al., (1998) Dev Biol 194: 114–128). Conversely, TGF-β1 is widely believed to inhibit satellite cell proliferation, as does contact with the myofiber plasmalemma, but not the basal lamina (Bischoff (1989); but see Hathaway et al. (1991) J. Cell Physiol. 146:435–441).
After muscle injury, satellite cells are activated and recruited to cycle as precursors for new muscle formation. Between injury and proliferation in vivo, satellite cells express immediate early genes after 3–6 hr., (Weiss, (1994) Acta Neuropathol. 87: 63–70; Kami, K., Noguchi, K., and Senba, E., (1995) Cell Tissue Res. 280: 11–19) and muscle regulatory genes after 6 hr. (Grounds, M. D., Garrett, K. L., Lai, M. C. Wright, W. E., and Bielharz, M. W. (1992) Cell Tissue Res. 267: 99–104) in concert with proliferating cell nuclear antigen (Chambers, R. L., and McDermott, J. C., (1996) Can. J. Appl. Physiol. 21: 155–184). The expression of these genes, release of growth factors like bFGF and DNA synthesis 24–30 hr. later are used to characterize muscle regeneration in injured and dystrophic muscle (Grounds, M. D., and McGeachie, J. K. (1989) Cell Tissue Res. 255: 385–391; Anderson, J. E., et al. (1995) Exp. Cell Res. 216: 325–334; Anderson, J. E. et al. (1998) Muscle Nerve 21: 1153–1165; Floss, T., Arnold, H.-H., and Braun, T., (1997) Genes Dev. 11: 2040–2051). The timing and sequence of events are specific to repair (Megeney, L. A., Kablar, B., Garrett, K., Anderson J. E., and Rudnicki, N. A., (1996) Genes Dev. 10: 1173–1183; Li, Z., Mericskay, M., Agbulut, O., Butler-Browne, G. Carlsson, L., Thronell, L. E., Babinet, C., and Paulin, D., (1997) J. Cell Biol. 139: 129–144; McIntosh, L. M., Garrett, K. L., Megeney L., Rudnicki, M. A., and Anderson, J. E., (1998b) Anat. Rec. 252: 311–324) although similar to development (Rudnicki, M. A., and Jaenisch, R., (1995) Bioessays 17: 203–209; Yun, K., and Wold, B. (1996) Current Opinion Cell Biol. 8: 877–889).
The fine structure of satellite cells, positioned intimately between the fiber sarcolemma and external lamina (Mauro, A. (1961) J. Biophys. Biochem. Cytol. 87: 225–251; Ishikawa, H. (1966) Z. Anat. Entwicklungsgesch 125: 43–63) changes during their transition from quiescence to activation. Nuclei enlarge and become euchromatic. The typical attenuated organelle-poor cytoplasm expands and organelles such as mitochondria and rough endoplasmic reticulum hypertrophy (Schultz (1976) Am. J. Anat. 147: 49–70; Snow (1977) Cell Tissue Res. 185, 399–408; Schultz et al. (1978) J. Exp. Zool. 206: 451–456; Schultz et al. (1985) Muscle Nerve 8: 217–222). However, while activation is recognised as essential to repair and defined as precursor stimulation and recruitment to cycle (Bischoff, R. (1990a). J. Cell Biol. 111: 201–207), the initial signal, timing and character of activation are not known (Schultz and McCormick (1994) Rev. Physiol Biochem. Pharmacol. 123: 213–257).
To date, the earliest indicator of satellite cell transformation during activation is the co-localization of hepatocyte growth factor (also called scatter factor, HGF/SF) with its receptor c-met shortly after injury in normal rat muscle (Tatsumi et al. (1998) Dev. Biol. 194: 114–128). In normal and regenerating muscle, satellite cells express c-met (Cornelison and Wold (1997) Dev. Biol. 19: 270–283; Tatsumi et al. (1998) Dev. Biol. 194: 114–128) and m-cadherin (Moore and Walsh (1993) Development 110: 1409–1420; Irinchev et al. (1994) Dev. Dynamics 199: 326–337; Rose et al. (1994) Dev. Dynamics 201: 245–259). While HGF/SF also plays a role in differentiation (Gal-Levi et al. (1998) Biochim. Biophys. Acta. 1402: 39–51.), it is the activating agent in extracts from crushed muscle (Tatsumi et al. (1998) Dev. Biol. 194: 114–128). Thus, the shift of HGF/SF from the periphery of the intact fiber to satellite cells means that activation follows soon after muscle damage.
Other observations indicate that the activation signal is transmitted along fibers from the site of direct injury. After segmental damage, satellite cells proliferate and fuse to form new myotubes both adjacent to the injury (Grounds and McGeachie (1987) Cell Tissue Res. 250: 563–569) and also at some distance from the injury near the ends of fibers (Klein-Ogus and Harris (1983) Cell Tissue Res. 230: 671–676; Schultz et al. (1985) Muscle Nerve 8: 217–222; Bischoff (1990) Development 109: 943–952; Grounds et al. (1992) Cell Tissue Res. 267: 99–104; McIntosh et al. (1994) Muscle Nerve 17: 444–453; McIntosh and Anderson (1995) Biochem. Cell Biol. 73: 181–190). Satellite cells are activated without trauma and make DNA after exercise, training, stretch, cold, compression, hypertrophy, suspension and denervation (Bischoff (1986a) Dev. Biol. 115: 140–147; Bischoff (1986) Dev. Biol. 111: 129–139; Bischoff (1990b) Development 109: 943–952; Darr and Schultz: (1987) J. Appl. Physiol. 63: 1816–1821; Darr and Schultz (1989) J. Appl. Physiol. 67: 1827–1834; Appell et al. (1988) Int. J. Sports Med. 9: 297–299; White and Esser (1989) Med. Sci. Sports Exerc. 21: S158–S16; Snow (1990) Anat. Rec. 227: 437–446; Winchester et al. (1991) Am. J. Physiol 260 (Cell Physiol 29): C206–C212; Buonanno et al. (1992) Nucleic Acids Res. 20: 539–544; Always (1997) J. Gerontol. A. Biol. Sci. Med. Sci. 52: B203–B211). Therefore, multiple signals initiate or mediate activation. Nonetheless, it is clear that DNA synthesis some 24–30 hr after injury is a delayed index of prior and completed satellite cell activation.
From the above description of the art, it is clear that muscle repair and formation are enabled by satellite cell activation and recruitment to cycle. However, the immediate chemical signal that triggers such activation and recruitment has not been identified. Consequently, current treatments aimed at improving muscle repair and formation in both normal and disease states have been limited to physical therapy treatments, non-specific treatments, such as the use of hormones, that affect multiple metabolic systems, treatments which involve transplantation of muscle cells, and treatments based on gene therapy. Other treatments to improve the state of muscle health have been directed to the modulation of muscle contraction (U.S. Pat. No. 5,583,101), rather than to the crucial initial activation events that enable the regeneration of healthy muscle. Notably, transplantation and gene therapy treatments are both at early experimental stages and their use requires a high level of expertise to perform.
One treatment aimed at muscle repair and formation is the use of hormones. It is known in the art that growth hormones promote increase in muscle mass. Such hormones (including the class of anabolic androgenic steroids) have been used in farm animals under experimental conditions. Glucocorticoids (e.g. deflazacort and prednisone) have been prescribed to Duchenne muscular dystrophy patients. However, because hormones tend to be involved in multiple physiological processes, their beneficial effects are often accompanied by many dose-limiting side-effects. The glucocorticoids, in particular, possess anti-inflammatory and immunomodulatory activities. The major side effects of the glucocorticoids are hypertension, peptic ulcers, increased susceptibility to infections, osteoporosis, hyperglycemia, and vascular occlusion (WO97/41144).
Another treatment aimed at muscle repair and formation involves the transfer of muscle cells (myoblasts) to the injured site. Autologous mouse skeletal muscle cells have been explanted from a healthy muscle, proliferated in vitro, and then implanted into a necrotized skeletal muscle site (Alameddine and Fardeau (1989) in Myoblast Transfer Therapy, Griggs and Karpati, eds., pp. 159–166). It was shown that the transplanted satellite cells were able to populate the necrotized area and differentiate into functional myotubes which then mature into fully functional myofibers. Similarly, PCT Publication WO 96/28541 discloses that histocompatible donor mouse myoblasts can be implanted into the weakened muscle of a mouse model of muscular dystrophy and differentiate into myofibers. In addition, it is shown that growth of the myoblasts in bFGF results in significantly more new myofibers at the implant site. In humans, clinical trials of myoblast transplantation have had limited or disappointing results (Karpati, G. et al. Clin. Genet (1999) 55: 1–8). Thus, skeletal muscle satellite cells, proliferated in vitro, may be able to serve as a source of myogenic precursor cells for muscle restoration or regeneration therapy.
The ability of skeletal muscle satellite cells to restore or regenerate injured skeletal muscle has led some researchers to test whether myogenic precursor cells could be used to replace lost or damaged myocardial muscle. For example, mouse fetal cardiomyocytes, which are not terminally differentiated and retain the ability to divide, have been directly injected into the myocardium of a syngeneic adult mouse, and have been shown to form new and apparently functional myocardium (Soonpaa et al. (1994) Science 264: 98–101). Significantly, it has been shown that skeletal muscle satellite cells, explanted from adult canine skeletal muscle can be proliferated in vitro and implanted into a site of myocardial cryoinjury, where they appear to differentiate into “cardiac-like” muscle cells, possibly in response to morphogenic signals present in the myocardium (Chiu et al. (1995) Ann. Thorac. Surg. 60:12–18).
Although myoblast transfer is a promising treatment, it is still not effective in restoring adequate numbers of functional fibers to a diseased muscle. Transfer of myoblasts collected from a donor and amplified in culture may allow sufficient quantity, but involves immune rejection and suppression problems. Morphogens that induce proliferation of myogenic precursor cells have been used to treat damage to the myocardium (WO 98/27995).
As an alternative treatment to cell-based therapies to deliver dystrophin, gene therapy has been used to provide, on an experimental basis, the active counterpart of the missing or mutated protein to the muscle precursor cells prior to injection of the precursor cells into the muscle (e.g. WO 91/12329). However, gene transfer in mammals has only limited success due to low level expression of the therapeutic protein in vivo (Partridge et al. (1991) Muscle Nerve 14: 197–212; Partridge et al. Nature Medicine (1998) 4: 1208–1209), difficulties with delivery of the gene to targetted myogenic cells (Feero WG et al. (1997) Gene Therapy 4: 664–674), and immune responses (Partridge TA. Myoblast transplantation. In: Lanza RP et al., (eds) Yearbook of Cell and Tissue Transplantation 1996/1997. Kluwer Academic Publications (1996) p 53–59, Netherlands; Guerette B. et al. (1997) Cell Transplantation 6: 101–107).
Accordingly, there exists a need for treatments that enable muscle repair and formation based on the innate ability of muscle to regenerate new muscle after injury.